Glutamine (Gln) is one of the twenty amino acids encoded by the standard genetic code. The glutamine side chain is an amide; it is formed by replacing a side-chain hydroxyl of glutamic acid with an amine functional group.
Glutamine is the most abundant free amino acid in the blood stream with concentrations in the range of 500 to 750 μM (Walsh et al., 1998). Yet, the majority of dietary glutamine does not enter the blood stream under normal conditions. This is because most of it is utilised as respiratory fuel by the epithelial cells in the small intestines. Both the lungs and the brain produce small amounts of glutamine (Newsholme, 2001). Skeletal muscles produce the majority of glutamine in the body, as they contain over 90% of the total glutamine (Newsholme, 2001).
Glutamine has a diverse range of functions in the human body. It is involved in nitrogen transport in the brain (Young & Ajami, 2001). Glutamine has a key role in the immune system. Lymphocyte proliferation is dependent on extracellular glutamine levels. Studies have also shown that macrophage activity is suppressed when glutamine levels are lowered. Glutamine is a key fuel source for immune cells (Newsholme, 2001).
Glutamine is found in foods high in proteins, such as fish, red meat, beans, and dairy products. Glutamine supplements are used in weightlifting, bodybuilding, endurance and other sports, as well as by those who suffer from muscular cramps or pain, particularly elderly people. Glutamine is used mainly in the diet to replenish the body's stores of amino acids that have been used during workouts or everyday activities. Studies examining the effects of excessive consumption of glutamine have thus far proved inconclusive. Supplementation is normally considered beneficial because amino acid replenishment is required after prolonged periods of exercise. For example, where a workout or exercise requires use of amino acids, this creates a need for amino acid stores to be replenished. See, e.g., Castell & Newsholme, 1997. For this reason, glutamine is recommended during fasting, or for people who suffer from physical trauma, immune deficiencies, or cancer.
There is a significant body of evidence that links glutamine-enriched diets with improvement of intestinal conditions, for example, aiding maintenance of gut barrier function, addressing intestinal cell proliferation and differentiation, as well as generally reducing septic morbidity and the symptoms of irritable bowel syndrome. See, e.g., Wischmeyer, 2006. The cleansing properties of glutamine are thought to stem from the fact that the intestinal extraction rate of glutamine is higher than that for other amino acids. This was discovered by comparing plasma concentration within the gut between glutamine-enriched and non-glutamine-enriched diets. Glutamine is therefore thought to be a key component for alleviating problems in the gastrointestinal tract. Yet, the concentration of glutamine varies in different varieties of food, and it can be difficult to quantify the clinical benefit that is derived.
In addition, glutamine supplements can reduce healing time after operations. See, e.g., Morlion et al., 1998. Hospital waiting times after abdominal surgery have been reduced by providing parenteral nutrition regimens containing amounts of glutamine to patients. Clinical trials have revealed that patients on glutamine supplementation regimes show improved nitrogen balances, generation of cysteinyl-leukotrienes from polymorphonuclear neutrophil granulocytes, and improved postoperative lymphocyte recovery and intestinal permeability, in comparison to patients lacking glutamine supplementation. These noted benefits were observed without any concomitant side effects.
Plasma glutamine levels fall during times of stress such as sepsis, injury, burns, and premature birth. This is because in the inflammatory state the glutamine consumption by immune cells and other tissues increase. This outstrips the supply of glutamine and results in reduced levels of glutamine in the blood, immunological tissue, and muscles. If the plasma concentration falls below 400 μM, it is termed glutamine-deficient (Newsholme, 2001). Exercise also has an effect on plasma glutamine levels. Both high intensity training and endurance training have been shown to reduce the levels of glutamine in the blood (levels are usually lower than 500 μM) (Walsh et al., 1998). Glutamine levels in the blood plasma are consistently higher in patients with urea cycle disorders (Serrano et al., 2011).
It has been suggested that high levels of glutamine and alanine along with lower levels of arginine and citrulline in the blood plasma can be used to detect the urea cycle disorders N-acetylglutamate synthetase deficiency (NAGSD) and carbamylphosphate synthetase deficiency (CPSD) (Serrano et al., 2011). It has also been suggested that high levels of glutamine in blood plasma is a possible biomarker of OTCD, a urea cycle disorder that affects 1 in 14000 births (Trinh et al., 2003). Lower levels of glutamine in the blood also have the potential to be an indicator for overtraining syndrome (OTS). Studies have shown that glutamine levels are lower than the normal base line in athletes suffering from overtraining (Walsh et al., 1998). Thus, reduced glutamine in the blood serum may be considered as a biomarker of overtraining (Agostini et al., 2010; Petibois et al., 2002; McKenzie, 1999).
Very low levels of L-glutamine in the brain may be symptomatic of Alzheimer's disease (see, e.g., Chen & Herrup, 2012) or other neurodegenerative disorders (Tsuroka et al., 2013), while very low levels in serum or saliva are associated with rapidly growing tumours (Iketa et al., 2012; Tan et al., 2013). Very high levels of L-glutamine in urine may be symptomatic of aminoaciduria from muscle or tissue breakdown due to range of conditions, such as burns, surgery, wasting diseases, or infection. Moreover, altered levels of glutamine analogues in urine may be associated with various medical treatments or conditions, including metabolic and renal diseases.
For medical diagnostics and scientific research, glutamine levels are commonly assessed by mass spectrometry (see, e.g., Trinh et al., 2003; Darmaun, et al., 1985). There are glutamine measuring kits available from a range of suppliers (see, e.g., Sigma-Aldrich, Stock No. GLN-2; see also, Bioassay Systems, Catalogue No. EGLN-100). There are also published methods using an Escherichia coli based biosensor for detecting glutamine levels in plants (Tessaro et al., 2012). There are other published methods of measuring glutamine in a liquid sample using a membrane with immobilised glutaminase and glutamate oxidase (WO 88/10424).
Glutamine is also produced commercially for use in pharmaceuticals and nutritional supplements. Current estimated production of L-glutamine is over 2000 tons annually (Kusumoto, 2001). Several bacterial strains have been reported for glutamine production, including Brevibacterium flavin (Tsuchida et al., 1987) and Flavobacterium rigense (Nabe et al., 1981). However, the most commonly used strain for large scale amino acid production is aerobic actinomycete Corynebacterium glutamicum (Hermann, 2003).
C. glutamicum was discovered as a glutamate producer in the 1950s, with industrial production of L-glutamine starting in the late 1960s. The wild-type strain of C. glutamicum has high levels of endogenous L-glutamate as well as L-glutamine (L-gln) (Rehm and Burkovski, 2011). Both L-glutamate and L-glutamine can be excreted by certain optimized producer strains (Rehm and Burkovski, 2011; Kusumoto et al., 2001). In industrial production, C. glutamicum produces bulk quantities of L-glutamate and L-lysine, as well as smaller amounts of other amino acids, including L-glutamine (Hermann 2003).
During bacterial production, glutamate can be converted to L-glutamine under the right fermentation conditions. This includes the presence of the glnA gene, coding for glutamine synthetase I (GSI), which catalyses the reaction (Jakoby et al., 1997). The glnA gene is native to C. glutamicum, but is overexpressed in strains used for glutamine production. There is also an improved variant of this gene, termed glnA′, which was produced by site directed mutagenesis and includes the encoded mutation Y405F (Jakoby et al., 1999). The wild-type glnA gene is most active during nitrogen starvation and down regulated by high nitrogen levels, as it is involved in the nitrogen regulation pathway. In contrast, the variant is not down regulated in the presence of nitrogen.
For commercial production, different methods have been used to maximise and measure glutamine levels. According to one method, the amount of both L-glutamate and L-glutamine is increased by introducing a gene encoding haemoglobin that binds oxygen under hypoxic conditions, as the conversion to L-glutamine is a very high oxygen/energy reaction. The presence of the haemoglobin gene increases the production of both amino acids by 25-30% (Liu et al., 2008). According to this method, L-glutamate and L-glutamine are assayed by HPLC performed on the supernatant of the cells. The ratio of L-glutamine to L-glutamate is obtained, which is important for commercial production. With such production, L-glutamate is considered a by-product and can make downstream processing more difficult and expensive (Li et al., 2007).
According to another method, the activity of glutamine synthetase enzyme itself is assessed rather than the actual amount of L-glutamine being produced. This method includes in vitro assays to measure the activity of the glutamine synthetase enzyme by looking at NADH depletion (Wakisaka et al., 1990). Yet another method attempts to optimise L-glutamine production through optimization of levels of glucose versus (NH4)2SO4 levels. This method utilises an enzyme based assay and Biochemistry Analyzer YSI 2700 (Yellow Springs Instrument Co., USA; Li et al., 2007). Older methods for measuring glutamine have used quantitative paper chromatography (Tsuchida et al., 1987), or have assessed glutamine synthetase activity using glutamate together with an alternative substrate (e.g. hydroxylamine) in place of ammonia. In the case of hydroxylamine this yields an alternative coloured product, γ-glutamylhydroxamate (Nabe et al., 1981).
Glutamine analogues are also widely used in industry, scientific research, and medicine. Alanyl-glutamine is a more stable analogue of glutamine that is generally used in tissue culture. It is more heat stable and less ammoniagenic than glutamine (see Sigma Aldrich, Product Information Sheet for A8185). Other analogues of glutamine have been noted as potential anti-cancer compounds, including 6-diazo-5-oxo-L-norleucine (L-DON), azaserine, and acivicin (Ahluwalia, 1990; Szkudlifiski et al., 1990).
Given the importance of glutamine and glutamine analogues in industrial production, nutrition, medicine, and basic scientific research, there is significant interest in developing new methods for identifying and measuring these compounds.